RARE DISEASE
Acute flaccid myelitis
Acute flaccid myelitis
Acute flaccid myelitis
Drug discovery
169
papers
On emerging therapies
Overview
Acute flaccid myelitis (AFM) is a rare, polio-like neurologic emergency characterized by acute flaccid limb weakness, often following respiratory or febrile prodromes. Primarily affecting children, it involves gray matter spinal cord inflammation, with potential progression to respiratory failure. While enteroviruses (particularly EV-D68) show temporal associations, direct causality remains unproven. Diagnosis requires MRI demonstrating T2-hyperintense spinal cord lesions (91% specificity) and CSF pleocytosis (white blood cells >5/mm³) [5][7][12].
Categories: rare neurological diseases
Research Papers
169 drug discovery papers about Acute flaccid myelitis. Recent publications:
169 drug discovery papers about Acute flaccid myelitis. Recent publications:
categories:
Small molecules
antibodies
2026-08-26 | Impact of Immunotherapy Combinations on Clinical Outcomes in Children with Acute Flaccid Paralysis: A Multicenter Intensive Care Study
Objective: Acute flaccid paralysis (AFP) is a syndrome characterized by the rapid onset of limb weakness and decreased muscle tone, frequently accompanied by respiratory or bulbar involvement. Methods: This retrospective multicenter study evaluated the clinical characteristics, treatment modalities, intensive care processes, and outcomes of 68 children diagnosed with AFP who were admitted to pediatric intensive care units (PICUs) between January 1, 2018, and January 1, 2023. Results: The patients’ ages ranged from 1 month to 18 years (mean 7.9 ± 4.5 years), and 33 (48.5%) were male. The mean PICU stay was 15.4 ± 19.5 days, and the mean total hospital stay was 23.3 ± 21.1 days. Respiratory support was required in 42 patients (61.8%). All patients received intravenous immunoglobulin (IVIG). Therapeutic plasma exchange (TPE) was administered to 25 of 43 Guillain–Barré syndrome (GBS) patients, 7 of 11 transverse myelitis (TM) patients, and 11 of 14 acute disseminated encephalomyelitis (ADEM) patients. Among 14 intubated GBS patients, 5 received IVIG for ≤2 days and 9 for ≥4 days. The duration of invasive mechanical ventilation (MV) was significantly longer in patients receiving IVIG for ≥4 days (P=.044). Guillain–Barré syndrome patients treated with combined IVIG and TPE had longer PICU stays compared with those receiving IVIG alone (P=.001). Conclusion: Combination immunotherapies were associated with longer MV duration and PICU stay compared with monotherapy in AFP patients. In intubated GBS patients, administration of IVIG over 2 days was associated with a shorter duration of invasive MV.
2026-08-20 | EVALUATING THE MECHANISMS OF PATHOGENESIS AND ANTIBODY-MEDIATED PROTECTION IN ENTEROVIRUS D68-INDUCED ACUTE FLACCID MYELITIS
Enterovirus D68 (EV-D68) is a major cause of acute flaccid myelitis (AFM), a severe neurologic disease in children that can result in sudden paralysis and lifelong disability. Currently, no approved therapies specifically target EV-D68-associated AFM. Monoclonal antibodies (mAbs) represent a promising therapeutic strategy because they combine viral neutralization through the Fab region with immune-cell recruitment through the Fc region. However, the contribution of Fc effector function to protection against EV-D68-induced neurologic disease remains unclear. This study investigated whether Fc effector function enhances protection against AFM using the partially neutralizing mAb EV68-41. Wild-type EV68-41 (EV68-41-WT), which retains Fc effector function, was compared with an Fc-deficient LALAPG variant (EV68-41-LALAPG) in an animal model of EV-D68 infection. Survival and neurologic outcomes were assessed and compared with the potently neutralizing mAb EV68-228. No significant difference in survival was observed between EV68-41-WT and EV68-41-LALAPG, with both providing approximately 60% survival. However, EV68-41-LALAPG-treated mice exhibited higher peak neurologic scores, indicating more severe limb paralysis, compared with EV68-41-WT-treated mice. In contrast, both EV68-228 variants provided 100% survival and complete neurologic protection. These findings demonstrate that Fc effector function can reduce neurologic disease severity even when it does not significantly improve survival. Together, the results suggest that effective mAb therapies for EV-D68-associated AFM should combine potent viral neutralization with Fc-mediated immune recruitment, potentially improving neurologic outcomes and reducing the burden of childhood paralysis.
2026-06-03 | Progress in enterovirus D68-neutralizing antibodies and vaccines.
Enterovirus D68 (EV-D68) causes severe respiratory diseases and acute flaccid myelitis (AFM), for which no licensed vaccines or therapeutics are currently available. Neutralizing antibodies (nAbs) block infection via binding capsid epitopes. This review summarizes the EV-D68 antigenic structure, nAb mechanisms (receptor blockade and conformational disruption), therapeutic prospects of nAbs, and vaccine progress (inactivated, virus-like particle [VLP], and nucleic acid). Key challenges, like antigenic drift and blood-brain barrier (BBB) penetration, are also highlighted to guide countermeasure development.
2026-03-02 | Cervical Myelitis Masquerading as Guillain-Barré Syndrome: A Case of Acute Flaccid Tetraplegia With a Sensory Level at the Third Cervical Dermatome
Acute flaccid tetraplegia with areflexia and respiratory failure is frequently attributed to Guillain-Barré syndrome in emergency practice. However, high cervical inflammatory spinal cord lesions may present with a similar clinical picture, leading to potential diagnostic delays and inappropriate initial management. A 26-year-old woman presented with sudden interscapular pain followed within hours by rapidly progressive weakness of all four limbs and acute respiratory distress. Neurological examination revealed generalized areflexia, absence of pyramidal signs, and a clearly defined sensory level at the third cervical dermatome. Cerebrospinal fluid examination was normal. Urgent spinal magnetic resonance imaging demonstrated an intramedullary inflammatory lesion extending from the second to the sixth cervical vertebrae, consistent with longitudinal extensive transverse myelitis. Extensive investigations for infectious, autoimmune, and demyelinating causes were inconclusive, and specific antibody testing was negative, limiting etiological characterization. The patient was treated with high-dose corticosteroids, plasma exchange, and rituximab. Despite partial radiological improvement, the neurological outcome remained unfavorable, with persistent severe motor deficit at follow-up. The presence of a sensory level in a patient with acute flaccid tetraplegia is a critical clinical sign that should prompt immediate spinal imaging. Early recognition of cervical transverse myelitis is essential to initiate timely immunotherapy and may influence neurological prognosis.
2025-03-24 | EV68-228-N monoclonal antibody treatment halts progression of paralysis in a mouse model of EV-D68 induced acute flaccid myelitis.
In 2014, 2016, and 2018, infection with enterovirus D68 (EV-D68) was associated with outbreaks of a poliomyelitis-like paralytic syndrome, called acute flaccid myelitis (AFM). While only a small fraction of patients infected with EV-D68 developed AFM, this subgroup of patients does not typically seek treatment until after the onset of neurological symptoms. There are currently no approved human monoclonal antibody therapies or vaccines available for EV-D68. Here, we show that a monoclonal antibody, EV68-228-N, can quickly stop the progression of paralysis in a mouse model of AFM, even when treatment is initiated after the onset of paralysis. We found that EV68-228-N effectively halted the progression of paralysis when tested against both 2014 and 2016 EV-D68 isolates in an immunocompetent mouse model of AFM. All animal experiments were conducted in a blinded fashion. The IC50 of EV68-228-N against 2014 and 2016 EV-D68 isolates was confirmed in vitro to be less than 330 ng/mL, and EV68-228-N was found to be equally effective at neutralizing 2018 and 2022 viral isolates without any evidence of emerging resistance. We further show that, following infection with EV-D68, mice treated with EV68-228-N have more surviving motor neurons in the spinal cord's lumbar enlargement than control treated animals. Taken together, this work suggests that EV68-228-N treatment has the potential to halt the progression of paralysis in AFM patients who present at the clinic with neurologic symptoms and that EV68-228-N will retain neutralization potential against emerging EV-D68 isolates. Enterovirus D-68 (EV-D68) associated acute flaccid myelitis (AFM) is an emergent poliomyelitis-like illness occurring predominantly in children. There are currently no proven effective therapies. We describe the use of a human monoclonal antibody (EV68-228-N) in a murine model of EV-D68 AFM in which therapy prevents progression of paralysis even when treatment is instituted after onset of weakness. This study is registered with ClinicalTrials.gov as NCT06444048.
small molecules
2026-09-25 | Development of Enterovirus D68 3C Protease Inhibitors with In Vivo Efficacy in a Neurological Infection Model
Abstract Enterovirus D68 (EV-D68) is a non-polio enterovirus that primarily causes respiratory illness but can also lead to severe disease, including acute flaccid myelitis. No approved vaccine or antiviral is available. Here, we report the rational design and structure−property optimization of dipeptidyl EV-D68 3C protease (3Cpro) inhibitors. Structure-guided optimization and prodrug modification identified Jun13133 as a potent antiviral active against multiple contemporary EV-D68 isolates and EV-A71, with high selectivity. Computational modeling supported a stable substrate-mimetic binding mode consistent with reported covalent 3Cpro inhibitors. Jun13133 also showed favorable in vivo pharmacokinetic properties. In a neonatal mouse model of EV-D68-induced neurological disease, Jun13133 mitigated paralysis, prevented weight loss, and reduced viral replication in spinal cord and muscle tissues. These results validate EV-D68 3Cpro as a druggable antiviral target and support Jun13133 as a promising lead for further development.
2026-09-21 | Preclinical evaluation of GW406108X, a broad-spectrum therapeutic blocking ULK1/2-mediated autophagy to inhibit enterovirus replication.
Enterovirus D68 (EV-D68) is an emerging respiratory pathogen, with severe cases developing into acute flaccid myelitis, a paralytic condition. Despite growing concern, no approved antivirals currently exist for EV-D68, underscoring the urgent need for therapeutic discovery. Here, we report the development of a high-throughput, phenotypic-based assay to screen 7986 compounds across nine diverse compound libraries for EV-D68 antivirals, on two EV-D68-susceptible cell lines (RD and H1299). The screen identified GW406108X as a potent post-entry inhibitor of EV-D68 infection (EC50: 1.804 μmol/L). Mechanistic studies using luciferase replicon assays, siRNA knockdowns, and drug-resistant mutant generation suggest that GW406108X targets the autophagy pathway through ULK1/2 inhibition. Transmission electron microscopy and fluorescence bioimaging demonstrate a significant reduction in autophagosome formation in treated, infected cells. This disruption likely impairs the virus's ability to exploit autophagy, which may in turn hinder replication organelle formation and non-lytic virion release, leading to reduced viral replication. Preclinical evaluations showed that GW406108X demonstrates strong antiviral efficacy without detectable cytotoxicity. These findings reveal a previously uncharacterized antiviral mechanism and position GW406108X as a promising candidate for antiviral therapeutic development. Findings from this study expand the current antiviral landscape for enteroviruses and represent a significant step toward clinical intervention for EV-D68 and potentially related viral pathogens.
2026-07-10 | Discovery of non-nucleoside inhibitors of the enterovirus D68 3D polymerase through crystallographic fragment and high-throughput biochemical screening
Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3D pol ) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3D pol , including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3D pol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3D pol , including hit-to-lead compound 727590, which displayed an IC 50 value of 25 µM and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses.
2026-06-18 | Reviewer #3 (Public review): Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity
This valuable study examines the cleavage of motor neuron nucleoporins by proteases of enterovirus D68, a pathogen associated with acute flaccid myelitis.The evidence supporting the effects of EV-D68 proteases on nuclear import and export is generally solid, as is the independent examination of EV-D68 protease on spinal cord neuron toxicity.The specific conclusions related to RNA export were considered overstated relative to the data presented.Abstract Enterovirus D68 (EV-D68) is an important pathogen associated with acute flaccid myelitis (AFM).The pathogenesis of AFM involves infection of spinal motor neurons and motor neuron death; however, the mechanisms linking EV-D68 infection to selective neurotoxicity are not well understood.Dysfunction of the nuclear pore complex (NPC) has been implicated in motor neuron injury in neurodegenerative diseases such as amyotrophic lateral sclerosis, and the NPC is also modified by picornavirus proteases during infection.We therefore sought to determine the impact of EV-D68 proteases on NPC composition and function.We demonstrate widespread disruption of NPC composition by EV-D68 2A and 3C proteases via direct cleavage of a relatively small number of nucleoporins, notably Nup98 and POM121, by 2A pro .Using reporter systems, we demonstrate that 2A pro inhibits nuclear transport of protein cargoes and disrupts the permeability barrier of the NPC, while having no apparent effect on RNA export.Independently, we show 2A pro is toxic to induced pluripotent stem cell-derived motor neurons by demonstrating a rescue of toxicity with the 2A pro inhibitor telaprevir at concentrations insufficient to inhibit viral replication.These findings expand our understanding of EV-D68 neuropathogenesis and provide a rationale for studying the NPC or 2A pro as therapeutic targets in AFM.
2026-05-21 | Author response: Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity
The picornavirus Enterovirus D68 (EV-D68) is an important pathogen associated with acute flaccid myelitis (AFM). The pathogenesis of AFM involves infection of spinal motor neurons and motor neuron death, however the mechanisms linking EV-D68 infection to selective neurotoxicity are not well understood. Dysfunction of the nuclear pore complex (NPC) has been implicated in motor neuron injury in neurodegenerative diseases such as amyotrophic lateral sclerosis, and the NPC is also modified by picornavirus proteases during the course of infection. We therefore sought to determine the impact of EV-D68 proteases on NPC composition and function. We demonstrate widespread disruption of NPC composition by EV-D68 2A and 3C proteases via the direct cleavage of a relatively small number of nucleoporins, notably Nup98 and POM121 by 2Apro. Using reporter systems, we demonstrate that 2Apro inhibits nuclear import and export of protein cargoes and also disrupts the permeability barrier of the NPC, while having no apparent effect on RNA export. Independently, we show that 2Apro is toxic to induced pluripotent stem cell derived motor neurons by demonstrating a rescue of toxicity with 2Apro inhibitor telaprevir at concentrations that are insufficient to inhibit viral replication. These findings expand our understanding of EV-D68 neuropathogenesis and provides a rationale for studying the NPC or 2Apro as therapeutic targets in AFM.
proteins
2026-08-27 | Seroepidemiology of Enterovirus D68 in Xiamen Children: A Cross-Sectional Study in 2022.
Enterovirus D68 (EV-D68) is a re-emerging pathogen associated with severe acute flaccid myelitis (AFM) and pneumonia, predominantly in children but also capable of causing severe disease in immunocompromised adults. Seroprevalence data are essential for understanding population immunity, age-specific susceptibility, and viral transmission dynamics; yet, such data for the period after the COVID-19 pandemic are scarce. We conducted a cross-sectional serological survey to characterize the neutralizing antibody profile of EV-D68 among children in Xiamen, China, in 2022. A total of 453 children aged 0-16 years (217 with acute respiratory tract infection [ARTI] and 236 without) were enrolled. Neutralizing antibody (NtAb) titers against the EV-D68 STL strain were measured using a microneutralization assay, seropositivity was defined as a titer ≥1:16. Multivariable regression and analysis of covariance were used to adjust for age and sex. Overall seroprevalence was 96.0% (435/453), exceeding 90.0% in every age subgroup (0-1 y: 100%; 1-3 y: 97.6%; 3-5 y: 92.6%; ≥5 y: 96.2%). The geometric mean titer (GMT) was 76.78 (95% CI: 68.32-85.24). After adjustment, ARTI status was not significantly associated with seropositivity (adjusted OR = 1.42, 95% CI: 0.46-4.41, p = 0.542). In an exploratory comparison, children with ARTI showed nominally higher antibody titers than those without (Mann-Whitney U test, p = 0.003), although the absence of EV-D68-specific PCR testing precludes etiologic attribution of respiratory symptoms to EV-D68 infection. Infants < 1 year showed the highest GMT (86.31), while children aged 1-3 years had the lowest GMT (63.52) and the largest low-titer fraction, indicating a susceptibility gap. Our study revealed EV-D68 circulated endemically in this pediatric population, establishing a high population immune baseline (>90.0%) with distinct age-dependent patterns. These findings provide key reference data for ongoing serosurveillance, outbreak risk assessment, and the evaluation of future vaccines or immunoprophylactic strategies should they become available.
2026-04-22 | Enterovirus-induced cleavage of Mitofusin 2 generates mitophagosomes for enveloped virion release.
Enterovirus D68 (EV-D68) is a plus-strand RNA virus that primarily causes respiratory infections in infants but, in rare cases, has been associated with the pediatric paralytic disease acute flaccid myelitis. We previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that the 3C protease of EV-D68 cleaves the mitochondrial fusion protein Mitofusin 2 near its C-terminal HR2 domain, inducing fragmentation of the mitochondrial network. This, in turn, triggers the formation of mitophagosomes, a hallmark of mitophagy, a selective form of autophagy that recycles mitochondria. Multiple hallmarks of mitophagy are observed during infection, including loss of mitochondrial membrane potential and Parkin translocation to the mitochondria, but mitochondrial degradation is blocked during infection. While autophagy plays multiple roles in enterovirus infection, depleting Mitofusin 2 or transiently overexpressing Mitofusin 2, particularly the cleavage-resistant mutant, specifically reduces EV-D68 release from cells without affecting intracellular titers. Our results show that enteroviruses induce mitophagosomes as vectors for nonlytic release of virions from cells.
2025-12-30 | Enterovirus D68 receptor usage: from static attachment to dynamic entry.
Enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen of notable clinical concern due to its association with severe respiratory disease and the paralytic complication acute flaccid myelitis (AFM). Viral tropism and pathogenesis are critically dictated by interactions with host cell receptors. Our understanding of this process has evolved from a simple model of sialic acid dependence to a dynamic paradigm involving a repertoire of attachment factors and proteinaceous entry receptors. This review synthesizes the evolving landscape of EV-D68 receptor usage. We detail the well-established role of α2,6-linked sialic acid as an attachment factor and uncoating trigger for historical strains. We further discuss the discovery of intracellular adhesion molecule-5 (ICAM-5) as a neuron-specific receptor that provides a molecular explanation for neurotropism in AFM. A pivotal recent advance is the identification of major facilitator superfamily domain-containing 6 (MFSD6) as an essential entry receptor for a broad range of EV-D68 strains in both respiratory and neuronal cells. We explore the implications of this receptor versatility, whereby the virus can switch between or co-opt sialic acid, ICAM-5, and MFSD6, a plasticity that influences tissue tropism and viral evolution. Finally, we highlight how these mechanistic insights, particularly the characterization of the MFSD6 interface, are paving the way for novel therapeutic strategies, such as engineered decoy receptors, and outline key future directions in the field.
2025-11-17 | A case of enterovirus D68-associated acute flaccid myelitis in an immunocompromised adult, Australia.
Acute flaccid myelitis (AFM) is a predominantly pediatric, polio-like syndrome associated with enterovirus D68. AFM in adults is rare, reported predominantly in immunocompromised hosts. Here, we report detection of enterovirus D68 in an immunocompromised Australian adult, specifically the B3 subgroup. This strain is phylogenetically linked to the 2018 AFM outbreaks in the USA and to documented cases in 2022 in France.
2025-04-28 | EV-D68 cleaves LARP1 and PABPC1 by 3Cpro to redirect host mRNA translation machinery toward its genomic RNA.
Enterovirus D68 (EV-D68) is an emerging pathogen associated with severe respiratory diseases and neurological complications, such as acute flaccid myelitis. EV-D68 has developed sophisticated mechanisms to hijack host translation machinery, facilitating its replication and impairing host mRNA translation. In this study, we demonstrate that EV-D68 cleaves La-related protein 1 (LARP1) and poly(A)-binding protein cytoplasmic 1 (PABPC1) through its proteases 3Cpro and 2Apro. Our results indicate that overexpressing LARP1 and PABPC1 significantly inhibits EV-D68 replication and reduces the virus-mediated suppression of host translation. While both LARP1 and PABPC1 regulate translation, they exert antiviral effects through distinct mechanisms. We found that LARP1 interacts with the 5'UTR of EV-D68 RNA through its LAM domain, and this interaction is crucial for its antiviral function. LARP1 translation modulation is also influenced by the mTOR and CDK1 signaling pathways. Viral infection inhibits mTOR and CDK1 phosphorylation, which enhances LARP1's binding to viral RNA and inhibits viral translation. To counteract this inhibition, EV-D68 cleaves LARP1 through 3Cpro, thereby promoting efficient viral translation. We also investigated other enteroviruses, such as EV-A71 and CV-A16, which similarly target LARP1 and PABPC1, indicating a conserved mechanism across enteroviruses. Our findings offer new insights into how EV-D68 manipulates host translation and highlight the potential of targeting LARP1 and PABPC1 for antiviral interventions.
vaccines
2026-07-28 | Boosting Neonatal Immunity: Zymosan-Adjuvanted Intranasal Bivalent Vaccine Shields Against Lethal EV-A71 and EV-D68 Infections 2246328
Abstract Introduction Non-polio enteroviruses, particularly EV-D68 and EV-A71, have emerged as major pediatric threats due to their association with acute flaccid myelitis and high mortality. While intranasal vaccines offer a promising route to block viral entry, their effectiveness is limited by poor induction of protective immunity. Zymosan, a yeast cell wall component, has shown its adjuvant potential in multiple vaccine models. This study aims to develop an effective intranasal vaccine incorporating zymosan to protect against EV-D68 and EV-A71 infections. Methods We first prepared an inactivated EV-D68 vaccine to compare immunogenicity across administration routes. Next, we tested monovalent and bivalent intranasal vaccines with zymosan to assess multi-dose and adjuvant effects. Finally, we evaluated vaccine efficacy in a neonatal mouse model. Results Two doses of inactivated EV-D68 with intranasal priming effectively induced mucosal and systemic immunity. A three-dose regimen further strengthened vaccine-specific immunity, with zymosan amplifying responses in both formulations and enhancing serum neutralization. Serum transfer from the zymosan-adjuvanted triple-vaccinated donors afforded significant protection against both viral challenges. Conclusion These findings demonstrated that combining bivalent antigens with zymosan broadened and enhanced the intranasal vaccine efficacy, providing a promising approach for early-life protection against EV-A71 and EV-D68 infections and advancing nasal spray vaccine strategies. Funding Source National Taiwan University Hospital Topic Categories Vaccines and Immunotherapy (VAC)
2026-06-15 | Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68.
Enterovirus D68 (EV-D68) primarily causes respiratory illnesses and has been implicated in acute flaccid myelitis. Although virus-like particle (VLP) and traditional inactivated whole-virion (IWV) vaccines have demonstrated efficacy in mice, their immunological differences remain undetermined. Here, we directly compared the immunogenic and structural properties of VLP and IWV vaccines derived from the same EV-D68 strain under identical conditions. Although VLP induced significantly lower levels of EV-D68-specific IgG than IWV, neutralizing antibody titers and protective effects against viral challenge were comparable between the two groups in mice. Passive transfer experiments in neonatal mice further confirmed protection against lethal infection for both vaccine groups. Notably, in contrast to the IWV vaccine, the VLP vaccine elicited antibodies that preferentially recognized a limited subset of epitopes. Cryo-electron microscopy analyses revealed that VLPs structurally resemble the native virus but display distinct features in regions corresponding to epitopes that show differential antibody reactivity between VLP and IWV vaccines. By integrating structural and immunological analyses, we established a mechanistic framework linking capsid architecture to vaccine-induced antibody specificity. These findings suggest that VLP is a promising EV-D68 vaccine antigen with distinct epitope recognition profiles driven by structural characteristics.
2026-06-03 | Neutralizing antibodies elicited in nonhuman primates by an enterovirus D68 virus-like particle vaccine target receptor binding sites.
Enterovirus D68 (EV-D68) is a picornavirus that causes biennial outbreaks of respiratory disease in young children that can progress to rare but severe complications, including acute flaccid myelitis (AFM). EV-D68 virus-like particles (VLPs) elicit potent neutralizing antibodies that are protective in animal models. Here, we report the isolation and characterization of monoclonal antibodies elicited by EV-D68 VLPs in nonhuman primates (NHPs). We identified five potently neutralizing mAbs targeting overlapping epitopes near the capsid fivefold axis of symmetry formed by pentamers of viral protein 1. Cryo-electron microscopy structures of mAbs 1E11 and 5H03 in complex with VLP revealed epitopes on the capsid that bridge the sialic acid binding site and the proteinaceous entry receptor major facilitator superfamily domain-containing protein 6 binding site. We further characterized the mechanisms by which these mAbs neutralize EV-D68 and found that mAbs can disrupt multiple steps in the EV-D68 life cycle, including promoting premature uncoating. Antibodies elicited by VLP immunization of NHP protected as well as a best-in-class human mAb in a mouse challenge model, although single-amino acid mutations in the capsid enabled viral escape. Our results demonstrate that EV-D68 VLP-elicited mAbs target major viral sites of vulnerability, provide insight into their mechanisms of neutralization, and reinforce the potential for VLP-based vaccines as a countermeasure for EV-D68.
2026-05-06 | Propagation and immunological characterization of three enterovirus D68 strains using serum-free HEK293A suspension cell culture.
Enterovirus D68 (EV-D68) is an emerging pathogen that typically causes respiratory illness but can also lead to acute flaccid myelitis, which has been reported in several outbreaks worldwide. As EV-D68 has been found to be difficult to infect and proliferate in the standard Vero cells, a viable manufacturing process for the development of inactivated EV-D68 vaccine is urgently needed. We performed cell line screening and found that the HEK293A is a suitable host for EV-D68 propagation. The EV-D68 TW2014 (B3) strain was used to develop the EV-D68 production using a serum-free HEK293A (sfHEK293A) suspension system. Two major EV-D68 particles, the empty and full viral particles, were separated by ultracentrifugation and were found to induce low and high neutralizing antibody responses, respectively, in animal studies. To facilitate the mass production of vaccine, a combination of size-exclusion and anion-exchange chromatography was developed to purify EV-D68 particles. High quality, pure EV-D68 particles could be obtained by this simple procedure for vaccine production. Additionally, we found that the EV-D68 MO (B1) and KY (A2) strains could also be propagated in this sfHEK293A cell system, but at a lower 33 °C condition. These three formalin-inactivated EV-D68s were shown to induce EV-D68-specific neutralizing responses and also neutralize a recently circulating strain, but failed to cross-neutralize enterovirus A71 (EV-A71) in sera assay. The combination of EV-D68 and EV-A71 in one vaccination induced the neutralizing responses against both viruses. Our results showed that EV-D68 particles produced from the sfHEK293A suspension culture could be used to prepare EV-D68 vaccine candidates. Purifying the full EV-D68 particles for vaccine production is recommended for inducing a high, specific immune response. Combination of EV-D68 and EV-A71 is beneficial for the development of bivalent enterovirus vaccine.
2025-07-15 | Immunoinformatic Based Designing of Immune Boosting and Nonallergenic Multi-epitope Subunit Vaccine Against the Enterovirus D68.
Enterovirus D68 (EV-D68) is a non-enveloped, positive-sense, singlestranded RNA virus known for causing severe respiratory illnesses and its association with acute flaccid myelitis (AFM) in children. Despite its increasing public health significance, no vaccines or antiviral drugs are currently available for EV-D68. This study aimed to design an immune-boosting multi-epitope subunit vaccine against EV-D68 using advanced immunoinformatic and machine learning approaches. Capsid proteins VP1, VP2, and VP3 of EV-D68 were screened for immunogenic HTL, CTL, and B-cell epitopes to develop a non-allergenic, highly immunogenic multi-epitope vaccine. Predicted epitopes were subjected to 3D structural modeling and molecular dynamics simulations to validate folding and structural stability. Molecular docking and immune simulation techniques were employed to evaluate vaccine-TLR3 interactions and predict immune responses, respectively. Molecular docking analysis revealed strong binding affinities between the vaccine constructs and the TLR3 receptor, with scores of -299 kcal/mol, -361 kcal/mol, -258 kcal/mol, and -312 kcal/mol for VP1, VP2, VP3, and combined vaccine-TLR3 complexes. Molecular dynamic simulation and dissociation constant analyses confirmed the strength of these interactions, with binding free energies ranging from -57.75 kcal/mol to -101.35 kcal/mol. Codon adaptation index (CAI) values of 0.96 and GC content of ~69% supported the high expression potential of the vaccine constructs. Immune simulations demonstrated robust immune responses characterized by elevated IgG, IgM, cytokines, and interleukins, along with effective antigen clearance. The strong molecular interactions with TLR3 and simulated immune responses suggest that the designed vaccines can activate both innate and adaptive immunity. The high CAI and GC values support their expression feasibility in E. coli, enhancing prospects for production. This study provides a strong foundation for the development of a safe and effective EV-D68 vaccine, showcasing the potential of computational vaccine design.
other
2025-11-18 | Efficacy and Safety of Plasmapheresis in Children With Acute Transverse or Flaccid Myelitis, and Guillain-Barré Syndrome.
Acute transverse myelitis (ATM), acute flaccid myelitis with polio-like myeloradiculitis (AFM) and Guillain-Barré syndrome (GBS) are severe neuroimmune disorders associated with high morbidity. First-line treatments include corticosteroids and intravenous immunoglobulins (IVIg), but they may be insufficient. Therapeutic plasma exchange (TPE) and immunoadsorption (IA) are second- or third-line options in pediatrics, despite their recognized efficacy in adults. Data on pediatric use remain limited. This study aimed to evaluate the efficacy and safety of apheresis in children with ATM, GBS or AFM. We conducted a retrospective monocentric study at Necker University Hospital, including pediatric patients diagnosed with ATM, GBS or AFM who underwent TPE/IA between 2014 and 2024. Functional outcomes were assessed using the modified Rankin Scale (mRS) at five time points, from peak disease severity to last follow-up. Safety was evaluated based on severe adverse events. Among 23 children (6 years [Q1-Q3]), 20 received TPE and 3 IA. 17 of 23 patients (74%) showed significant improvement by the end of treatment. The median mRS was 5 (IQR 4-5) before apheresis, improving to 4 (IQR 2-4) at the end of TPE/IA, and decreasing to 2 at 6 months. Brainstem function fully recovered in all surviving patients. Among 14 children with sphincter dysfunction, 5 (36%) still required intermittent catheterization. No treatment discontinuations occurred, but two patients died, one from venous air embolism, one after life-sustaining treatment limitation. TPE and IA appear effective in pediatric ATM, AFM and GBS. Larger studies are needed to confirm long-term efficacy and refine treatment guidelines.
2024-03-23 | Transcription factor EB (TFEB) interaction with RagC is disrupted during Enterovirus D68 Infection
Abstract Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is essential for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells, but not in autophagy defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. Importance Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis (AFM). The latter disease is associated with multiple non-polio enterovirus species, including Enterovirus D68 (EV-D68), Enterovirus 71 (EV-71), and coxsackievirus B3 (CVB3). Here we demonstrate that EV-D68 interacts with a host transcription factor, TFEB, to promote vRNA replication and regulate egress of virions from cells. TFEB was previously implicated in viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here we show EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically-important viruses differently.
2024-02-29 | Human B cells and dendritic cells are susceptible and permissive to enterovirus D68 infection.
Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.
2023-09-04 | Contemporary enterovirus-D68 isolates infect human spinal cord organoids.
Enterovirus D68 (EV-D68) is a nonpolio enterovirus associated with severe respiratory illness and acute flaccid myelitis (AFM), a polio-like illness causing paralysis in children. AFM outbreaks have been associated with increased circulation and genetic diversity of EV-D68 since 2014, although the virus was discovered in the 1960s. The mechanisms by which EV-D68 targets the central nervous system are unknown. Since enteroviruses are human pathogens that do not routinely infect other animal species, establishment of a human model of the central nervous system is essential for understanding pathogenesis. Here, we describe two human spinal cord organoid (hSCO)-based models for EV-D68 infection derived from induced, pluripotent stem cell (iPSC) lines. One hSCO model consists primarily of spinal motor neurons, while the another model comprises multiple neuronal cell lineages, including motor neurons, interneurons, and glial cells. These hSCOs can be productively infected with contemporary strains, but not a historic strain, of EV-D68 and produce extracellular virus for at least 2 weeks without appreciable cytopathic effect. By comparison, infection with hSCO with another enterovirus, echovirus 11, causes significant structural destruction and apoptosis. Together, these findings suggest that EV-D68 infection is not the sole mediator of neuronal cell death in the spinal cord in those with AFM and that secondary injury from the immune response likely contributes to pathogenesis. IMPORTANCE AFM is a rare condition that causes significant morbidity in affected children, often contributing to life-long sequelae. It is unknown how EV-D68 causes paralysis in children, and effective therapeutic and preventative strategies are not available. Mice are not native hosts for EV-D68, and thus, existing mouse models use immunosuppressed or neonatal mice, mouse-adapted viruses, or intracranial inoculations. To complement existing models, we report two hSCO models for EV-D68 infection. These three-dimensional, multicellular models comprised human cells and include multiple neural lineages, including motor neurons, interneurons, and glial cells. These new hSCO models for EV-D68 infection will contribute to understanding how EV-D68 damages the human spinal cord, which could lead to new therapeutic and prophylactic strategies for this virus.
2023-01-20 | RNA Structure in the 5′ Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes
Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5′ UTR of the genome compared to the historical Fermon strain. The structure of the 5′ UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5′ UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5′ UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.
antibodies
2026-08-26 | Impact of Immunotherapy Combinations on Clinical Outcomes in Children with Acute Flaccid Paralysis: A Multicenter Intensive Care Study
Objective: Acute flaccid paralysis (AFP) is a syndrome characterized by the rapid onset of limb weakness and decreased muscle tone, frequently accompanied by respiratory or bulbar involvement. Methods: This retrospective multicenter study evaluated the clinical characteristics, treatment modalities, intensive care processes, and outcomes of 68 children diagnosed with AFP who were admitted to pediatric intensive care units (PICUs) between January 1, 2018, and January 1, 2023. Results: The patients’ ages ranged from 1 month to 18 years (mean 7.9 ± 4.5 years), and 33 (48.5%) were male. The mean PICU stay was 15.4 ± 19.5 days, and the mean total hospital stay was 23.3 ± 21.1 days. Respiratory support was required in 42 patients (61.8%). All patients received intravenous immunoglobulin (IVIG). Therapeutic plasma exchange (TPE) was administered to 25 of 43 Guillain–Barré syndrome (GBS) patients, 7 of 11 transverse myelitis (TM) patients, and 11 of 14 acute disseminated encephalomyelitis (ADEM) patients. Among 14 intubated GBS patients, 5 received IVIG for ≤2 days and 9 for ≥4 days. The duration of invasive mechanical ventilation (MV) was significantly longer in patients receiving IVIG for ≥4 days (P=.044). Guillain–Barré syndrome patients treated with combined IVIG and TPE had longer PICU stays compared with those receiving IVIG alone (P=.001). Conclusion: Combination immunotherapies were associated with longer MV duration and PICU stay compared with monotherapy in AFP patients. In intubated GBS patients, administration of IVIG over 2 days was associated with a shorter duration of invasive MV.
2026-08-20 | EVALUATING THE MECHANISMS OF PATHOGENESIS AND ANTIBODY-MEDIATED PROTECTION IN ENTEROVIRUS D68-INDUCED ACUTE FLACCID MYELITIS
Enterovirus D68 (EV-D68) is a major cause of acute flaccid myelitis (AFM), a severe neurologic disease in children that can result in sudden paralysis and lifelong disability. Currently, no approved therapies specifically target EV-D68-associated AFM. Monoclonal antibodies (mAbs) represent a promising therapeutic strategy because they combine viral neutralization through the Fab region with immune-cell recruitment through the Fc region. However, the contribution of Fc effector function to protection against EV-D68-induced neurologic disease remains unclear. This study investigated whether Fc effector function enhances protection against AFM using the partially neutralizing mAb EV68-41. Wild-type EV68-41 (EV68-41-WT), which retains Fc effector function, was compared with an Fc-deficient LALAPG variant (EV68-41-LALAPG) in an animal model of EV-D68 infection. Survival and neurologic outcomes were assessed and compared with the potently neutralizing mAb EV68-228. No significant difference in survival was observed between EV68-41-WT and EV68-41-LALAPG, with both providing approximately 60% survival. However, EV68-41-LALAPG-treated mice exhibited higher peak neurologic scores, indicating more severe limb paralysis, compared with EV68-41-WT-treated mice. In contrast, both EV68-228 variants provided 100% survival and complete neurologic protection. These findings demonstrate that Fc effector function can reduce neurologic disease severity even when it does not significantly improve survival. Together, the results suggest that effective mAb therapies for EV-D68-associated AFM should combine potent viral neutralization with Fc-mediated immune recruitment, potentially improving neurologic outcomes and reducing the burden of childhood paralysis.
2026-06-03 | Progress in enterovirus D68-neutralizing antibodies and vaccines.
Enterovirus D68 (EV-D68) causes severe respiratory diseases and acute flaccid myelitis (AFM), for which no licensed vaccines or therapeutics are currently available. Neutralizing antibodies (nAbs) block infection via binding capsid epitopes. This review summarizes the EV-D68 antigenic structure, nAb mechanisms (receptor blockade and conformational disruption), therapeutic prospects of nAbs, and vaccine progress (inactivated, virus-like particle [VLP], and nucleic acid). Key challenges, like antigenic drift and blood-brain barrier (BBB) penetration, are also highlighted to guide countermeasure development.
2026-03-02 | Cervical Myelitis Masquerading as Guillain-Barré Syndrome: A Case of Acute Flaccid Tetraplegia With a Sensory Level at the Third Cervical Dermatome
Acute flaccid tetraplegia with areflexia and respiratory failure is frequently attributed to Guillain-Barré syndrome in emergency practice. However, high cervical inflammatory spinal cord lesions may present with a similar clinical picture, leading to potential diagnostic delays and inappropriate initial management. A 26-year-old woman presented with sudden interscapular pain followed within hours by rapidly progressive weakness of all four limbs and acute respiratory distress. Neurological examination revealed generalized areflexia, absence of pyramidal signs, and a clearly defined sensory level at the third cervical dermatome. Cerebrospinal fluid examination was normal. Urgent spinal magnetic resonance imaging demonstrated an intramedullary inflammatory lesion extending from the second to the sixth cervical vertebrae, consistent with longitudinal extensive transverse myelitis. Extensive investigations for infectious, autoimmune, and demyelinating causes were inconclusive, and specific antibody testing was negative, limiting etiological characterization. The patient was treated with high-dose corticosteroids, plasma exchange, and rituximab. Despite partial radiological improvement, the neurological outcome remained unfavorable, with persistent severe motor deficit at follow-up. The presence of a sensory level in a patient with acute flaccid tetraplegia is a critical clinical sign that should prompt immediate spinal imaging. Early recognition of cervical transverse myelitis is essential to initiate timely immunotherapy and may influence neurological prognosis.
2025-03-24 | EV68-228-N monoclonal antibody treatment halts progression of paralysis in a mouse model of EV-D68 induced acute flaccid myelitis.
In 2014, 2016, and 2018, infection with enterovirus D68 (EV-D68) was associated with outbreaks of a poliomyelitis-like paralytic syndrome, called acute flaccid myelitis (AFM). While only a small fraction of patients infected with EV-D68 developed AFM, this subgroup of patients does not typically seek treatment until after the onset of neurological symptoms. There are currently no approved human monoclonal antibody therapies or vaccines available for EV-D68. Here, we show that a monoclonal antibody, EV68-228-N, can quickly stop the progression of paralysis in a mouse model of AFM, even when treatment is initiated after the onset of paralysis. We found that EV68-228-N effectively halted the progression of paralysis when tested against both 2014 and 2016 EV-D68 isolates in an immunocompetent mouse model of AFM. All animal experiments were conducted in a blinded fashion. The IC50 of EV68-228-N against 2014 and 2016 EV-D68 isolates was confirmed in vitro to be less than 330 ng/mL, and EV68-228-N was found to be equally effective at neutralizing 2018 and 2022 viral isolates without any evidence of emerging resistance. We further show that, following infection with EV-D68, mice treated with EV68-228-N have more surviving motor neurons in the spinal cord's lumbar enlargement than control treated animals. Taken together, this work suggests that EV68-228-N treatment has the potential to halt the progression of paralysis in AFM patients who present at the clinic with neurologic symptoms and that EV68-228-N will retain neutralization potential against emerging EV-D68 isolates. Enterovirus D-68 (EV-D68) associated acute flaccid myelitis (AFM) is an emergent poliomyelitis-like illness occurring predominantly in children. There are currently no proven effective therapies. We describe the use of a human monoclonal antibody (EV68-228-N) in a murine model of EV-D68 AFM in which therapy prevents progression of paralysis even when treatment is instituted after onset of weakness. This study is registered with ClinicalTrials.gov as NCT06444048.
small molecules
2026-09-25 | Development of Enterovirus D68 3C Protease Inhibitors with In Vivo Efficacy in a Neurological Infection Model
Abstract Enterovirus D68 (EV-D68) is a non-polio enterovirus that primarily causes respiratory illness but can also lead to severe disease, including acute flaccid myelitis. No approved vaccine or antiviral is available. Here, we report the rational design and structure−property optimization of dipeptidyl EV-D68 3C protease (3Cpro) inhibitors. Structure-guided optimization and prodrug modification identified Jun13133 as a potent antiviral active against multiple contemporary EV-D68 isolates and EV-A71, with high selectivity. Computational modeling supported a stable substrate-mimetic binding mode consistent with reported covalent 3Cpro inhibitors. Jun13133 also showed favorable in vivo pharmacokinetic properties. In a neonatal mouse model of EV-D68-induced neurological disease, Jun13133 mitigated paralysis, prevented weight loss, and reduced viral replication in spinal cord and muscle tissues. These results validate EV-D68 3Cpro as a druggable antiviral target and support Jun13133 as a promising lead for further development.
2026-09-21 | Preclinical evaluation of GW406108X, a broad-spectrum therapeutic blocking ULK1/2-mediated autophagy to inhibit enterovirus replication.
Enterovirus D68 (EV-D68) is an emerging respiratory pathogen, with severe cases developing into acute flaccid myelitis, a paralytic condition. Despite growing concern, no approved antivirals currently exist for EV-D68, underscoring the urgent need for therapeutic discovery. Here, we report the development of a high-throughput, phenotypic-based assay to screen 7986 compounds across nine diverse compound libraries for EV-D68 antivirals, on two EV-D68-susceptible cell lines (RD and H1299). The screen identified GW406108X as a potent post-entry inhibitor of EV-D68 infection (EC50: 1.804 μmol/L). Mechanistic studies using luciferase replicon assays, siRNA knockdowns, and drug-resistant mutant generation suggest that GW406108X targets the autophagy pathway through ULK1/2 inhibition. Transmission electron microscopy and fluorescence bioimaging demonstrate a significant reduction in autophagosome formation in treated, infected cells. This disruption likely impairs the virus's ability to exploit autophagy, which may in turn hinder replication organelle formation and non-lytic virion release, leading to reduced viral replication. Preclinical evaluations showed that GW406108X demonstrates strong antiviral efficacy without detectable cytotoxicity. These findings reveal a previously uncharacterized antiviral mechanism and position GW406108X as a promising candidate for antiviral therapeutic development. Findings from this study expand the current antiviral landscape for enteroviruses and represent a significant step toward clinical intervention for EV-D68 and potentially related viral pathogens.
2026-07-10 | Discovery of non-nucleoside inhibitors of the enterovirus D68 3D polymerase through crystallographic fragment and high-throughput biochemical screening
Enterovirus D68 (EV-D68) is a non-polio picornavirus that has caused increasing rates of severe respiratory illness and acute flaccid myelitis in children worldwide this century. There are no approved vaccines or antivirals for EV-D68. Thus, we conducted a crystallographic fragment screening (CFS) and a high-throughput screening (HTS) biochemical assay against the EV-D68 RNA-dependent RNA polymerase 3D (3D pol ) to identify ligandable sites and non-nucleoside compounds that can spearhead anti-enteroviral drug discovery. The CFS, involving 650 fragments, identified 68 hit compounds (~10% hit rate) distributed across 3D pol , including the functionally relevant sites RNA template channel, Active site, and RNA primer channel, and the previously unknown "Thumb site II" and "Index-middle finger pocket". Inhibition assays confirmed that compounds binding to each site can inhibit EV-D68 3D pol activity. The HTS, a fluorescence-based PicoGreen biochemical assay, permitted screening 50,000 compounds of the ChemBridge Premium Library (0.77% hit rate). After a second-round dose-response screening, we identified 5-aminoindazole as a promising scaffold that inhibits EV-D68 3D pol , including hit-to-lead compound 727590, which displayed an IC 50 value of 25 µM and preliminary structure-activity relationships. These hits offer amenable starting points for discovery and development of non-nucleoside inhibitors and provide opportunities for structure-based drug design against enteroviruses.
2026-06-18 | Reviewer #3 (Public review): Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity
This valuable study examines the cleavage of motor neuron nucleoporins by proteases of enterovirus D68, a pathogen associated with acute flaccid myelitis.The evidence supporting the effects of EV-D68 proteases on nuclear import and export is generally solid, as is the independent examination of EV-D68 protease on spinal cord neuron toxicity.The specific conclusions related to RNA export were considered overstated relative to the data presented.Abstract Enterovirus D68 (EV-D68) is an important pathogen associated with acute flaccid myelitis (AFM).The pathogenesis of AFM involves infection of spinal motor neurons and motor neuron death; however, the mechanisms linking EV-D68 infection to selective neurotoxicity are not well understood.Dysfunction of the nuclear pore complex (NPC) has been implicated in motor neuron injury in neurodegenerative diseases such as amyotrophic lateral sclerosis, and the NPC is also modified by picornavirus proteases during infection.We therefore sought to determine the impact of EV-D68 proteases on NPC composition and function.We demonstrate widespread disruption of NPC composition by EV-D68 2A and 3C proteases via direct cleavage of a relatively small number of nucleoporins, notably Nup98 and POM121, by 2A pro .Using reporter systems, we demonstrate that 2A pro inhibits nuclear transport of protein cargoes and disrupts the permeability barrier of the NPC, while having no apparent effect on RNA export.Independently, we show 2A pro is toxic to induced pluripotent stem cell-derived motor neurons by demonstrating a rescue of toxicity with the 2A pro inhibitor telaprevir at concentrations insufficient to inhibit viral replication.These findings expand our understanding of EV-D68 neuropathogenesis and provide a rationale for studying the NPC or 2A pro as therapeutic targets in AFM.
2026-05-21 | Author response: Enterovirus D68 2A protease causes nuclear pore complex dysfunction and independently contributes to motor neuron toxicity
The picornavirus Enterovirus D68 (EV-D68) is an important pathogen associated with acute flaccid myelitis (AFM). The pathogenesis of AFM involves infection of spinal motor neurons and motor neuron death, however the mechanisms linking EV-D68 infection to selective neurotoxicity are not well understood. Dysfunction of the nuclear pore complex (NPC) has been implicated in motor neuron injury in neurodegenerative diseases such as amyotrophic lateral sclerosis, and the NPC is also modified by picornavirus proteases during the course of infection. We therefore sought to determine the impact of EV-D68 proteases on NPC composition and function. We demonstrate widespread disruption of NPC composition by EV-D68 2A and 3C proteases via the direct cleavage of a relatively small number of nucleoporins, notably Nup98 and POM121 by 2Apro. Using reporter systems, we demonstrate that 2Apro inhibits nuclear import and export of protein cargoes and also disrupts the permeability barrier of the NPC, while having no apparent effect on RNA export. Independently, we show that 2Apro is toxic to induced pluripotent stem cell derived motor neurons by demonstrating a rescue of toxicity with 2Apro inhibitor telaprevir at concentrations that are insufficient to inhibit viral replication. These findings expand our understanding of EV-D68 neuropathogenesis and provides a rationale for studying the NPC or 2Apro as therapeutic targets in AFM.
proteins
2026-08-27 | Seroepidemiology of Enterovirus D68 in Xiamen Children: A Cross-Sectional Study in 2022.
Enterovirus D68 (EV-D68) is a re-emerging pathogen associated with severe acute flaccid myelitis (AFM) and pneumonia, predominantly in children but also capable of causing severe disease in immunocompromised adults. Seroprevalence data are essential for understanding population immunity, age-specific susceptibility, and viral transmission dynamics; yet, such data for the period after the COVID-19 pandemic are scarce. We conducted a cross-sectional serological survey to characterize the neutralizing antibody profile of EV-D68 among children in Xiamen, China, in 2022. A total of 453 children aged 0-16 years (217 with acute respiratory tract infection [ARTI] and 236 without) were enrolled. Neutralizing antibody (NtAb) titers against the EV-D68 STL strain were measured using a microneutralization assay, seropositivity was defined as a titer ≥1:16. Multivariable regression and analysis of covariance were used to adjust for age and sex. Overall seroprevalence was 96.0% (435/453), exceeding 90.0% in every age subgroup (0-1 y: 100%; 1-3 y: 97.6%; 3-5 y: 92.6%; ≥5 y: 96.2%). The geometric mean titer (GMT) was 76.78 (95% CI: 68.32-85.24). After adjustment, ARTI status was not significantly associated with seropositivity (adjusted OR = 1.42, 95% CI: 0.46-4.41, p = 0.542). In an exploratory comparison, children with ARTI showed nominally higher antibody titers than those without (Mann-Whitney U test, p = 0.003), although the absence of EV-D68-specific PCR testing precludes etiologic attribution of respiratory symptoms to EV-D68 infection. Infants < 1 year showed the highest GMT (86.31), while children aged 1-3 years had the lowest GMT (63.52) and the largest low-titer fraction, indicating a susceptibility gap. Our study revealed EV-D68 circulated endemically in this pediatric population, establishing a high population immune baseline (>90.0%) with distinct age-dependent patterns. These findings provide key reference data for ongoing serosurveillance, outbreak risk assessment, and the evaluation of future vaccines or immunoprophylactic strategies should they become available.
2026-04-22 | Enterovirus-induced cleavage of Mitofusin 2 generates mitophagosomes for enveloped virion release.
Enterovirus D68 (EV-D68) is a plus-strand RNA virus that primarily causes respiratory infections in infants but, in rare cases, has been associated with the pediatric paralytic disease acute flaccid myelitis. We previously demonstrated that EV-D68 induces nonselective autophagy for its benefit. Here, we demonstrate that the 3C protease of EV-D68 cleaves the mitochondrial fusion protein Mitofusin 2 near its C-terminal HR2 domain, inducing fragmentation of the mitochondrial network. This, in turn, triggers the formation of mitophagosomes, a hallmark of mitophagy, a selective form of autophagy that recycles mitochondria. Multiple hallmarks of mitophagy are observed during infection, including loss of mitochondrial membrane potential and Parkin translocation to the mitochondria, but mitochondrial degradation is blocked during infection. While autophagy plays multiple roles in enterovirus infection, depleting Mitofusin 2 or transiently overexpressing Mitofusin 2, particularly the cleavage-resistant mutant, specifically reduces EV-D68 release from cells without affecting intracellular titers. Our results show that enteroviruses induce mitophagosomes as vectors for nonlytic release of virions from cells.
2025-12-30 | Enterovirus D68 receptor usage: from static attachment to dynamic entry.
Enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen of notable clinical concern due to its association with severe respiratory disease and the paralytic complication acute flaccid myelitis (AFM). Viral tropism and pathogenesis are critically dictated by interactions with host cell receptors. Our understanding of this process has evolved from a simple model of sialic acid dependence to a dynamic paradigm involving a repertoire of attachment factors and proteinaceous entry receptors. This review synthesizes the evolving landscape of EV-D68 receptor usage. We detail the well-established role of α2,6-linked sialic acid as an attachment factor and uncoating trigger for historical strains. We further discuss the discovery of intracellular adhesion molecule-5 (ICAM-5) as a neuron-specific receptor that provides a molecular explanation for neurotropism in AFM. A pivotal recent advance is the identification of major facilitator superfamily domain-containing 6 (MFSD6) as an essential entry receptor for a broad range of EV-D68 strains in both respiratory and neuronal cells. We explore the implications of this receptor versatility, whereby the virus can switch between or co-opt sialic acid, ICAM-5, and MFSD6, a plasticity that influences tissue tropism and viral evolution. Finally, we highlight how these mechanistic insights, particularly the characterization of the MFSD6 interface, are paving the way for novel therapeutic strategies, such as engineered decoy receptors, and outline key future directions in the field.
2025-11-17 | A case of enterovirus D68-associated acute flaccid myelitis in an immunocompromised adult, Australia.
Acute flaccid myelitis (AFM) is a predominantly pediatric, polio-like syndrome associated with enterovirus D68. AFM in adults is rare, reported predominantly in immunocompromised hosts. Here, we report detection of enterovirus D68 in an immunocompromised Australian adult, specifically the B3 subgroup. This strain is phylogenetically linked to the 2018 AFM outbreaks in the USA and to documented cases in 2022 in France.
2025-04-28 | EV-D68 cleaves LARP1 and PABPC1 by 3Cpro to redirect host mRNA translation machinery toward its genomic RNA.
Enterovirus D68 (EV-D68) is an emerging pathogen associated with severe respiratory diseases and neurological complications, such as acute flaccid myelitis. EV-D68 has developed sophisticated mechanisms to hijack host translation machinery, facilitating its replication and impairing host mRNA translation. In this study, we demonstrate that EV-D68 cleaves La-related protein 1 (LARP1) and poly(A)-binding protein cytoplasmic 1 (PABPC1) through its proteases 3Cpro and 2Apro. Our results indicate that overexpressing LARP1 and PABPC1 significantly inhibits EV-D68 replication and reduces the virus-mediated suppression of host translation. While both LARP1 and PABPC1 regulate translation, they exert antiviral effects through distinct mechanisms. We found that LARP1 interacts with the 5'UTR of EV-D68 RNA through its LAM domain, and this interaction is crucial for its antiviral function. LARP1 translation modulation is also influenced by the mTOR and CDK1 signaling pathways. Viral infection inhibits mTOR and CDK1 phosphorylation, which enhances LARP1's binding to viral RNA and inhibits viral translation. To counteract this inhibition, EV-D68 cleaves LARP1 through 3Cpro, thereby promoting efficient viral translation. We also investigated other enteroviruses, such as EV-A71 and CV-A16, which similarly target LARP1 and PABPC1, indicating a conserved mechanism across enteroviruses. Our findings offer new insights into how EV-D68 manipulates host translation and highlight the potential of targeting LARP1 and PABPC1 for antiviral interventions.
vaccines
2026-07-28 | Boosting Neonatal Immunity: Zymosan-Adjuvanted Intranasal Bivalent Vaccine Shields Against Lethal EV-A71 and EV-D68 Infections 2246328
Abstract Introduction Non-polio enteroviruses, particularly EV-D68 and EV-A71, have emerged as major pediatric threats due to their association with acute flaccid myelitis and high mortality. While intranasal vaccines offer a promising route to block viral entry, their effectiveness is limited by poor induction of protective immunity. Zymosan, a yeast cell wall component, has shown its adjuvant potential in multiple vaccine models. This study aims to develop an effective intranasal vaccine incorporating zymosan to protect against EV-D68 and EV-A71 infections. Methods We first prepared an inactivated EV-D68 vaccine to compare immunogenicity across administration routes. Next, we tested monovalent and bivalent intranasal vaccines with zymosan to assess multi-dose and adjuvant effects. Finally, we evaluated vaccine efficacy in a neonatal mouse model. Results Two doses of inactivated EV-D68 with intranasal priming effectively induced mucosal and systemic immunity. A three-dose regimen further strengthened vaccine-specific immunity, with zymosan amplifying responses in both formulations and enhancing serum neutralization. Serum transfer from the zymosan-adjuvanted triple-vaccinated donors afforded significant protection against both viral challenges. Conclusion These findings demonstrated that combining bivalent antigens with zymosan broadened and enhanced the intranasal vaccine efficacy, providing a promising approach for early-life protection against EV-A71 and EV-D68 infections and advancing nasal spray vaccine strategies. Funding Source National Taiwan University Hospital Topic Categories Vaccines and Immunotherapy (VAC)
2026-06-15 | Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68.
Enterovirus D68 (EV-D68) primarily causes respiratory illnesses and has been implicated in acute flaccid myelitis. Although virus-like particle (VLP) and traditional inactivated whole-virion (IWV) vaccines have demonstrated efficacy in mice, their immunological differences remain undetermined. Here, we directly compared the immunogenic and structural properties of VLP and IWV vaccines derived from the same EV-D68 strain under identical conditions. Although VLP induced significantly lower levels of EV-D68-specific IgG than IWV, neutralizing antibody titers and protective effects against viral challenge were comparable between the two groups in mice. Passive transfer experiments in neonatal mice further confirmed protection against lethal infection for both vaccine groups. Notably, in contrast to the IWV vaccine, the VLP vaccine elicited antibodies that preferentially recognized a limited subset of epitopes. Cryo-electron microscopy analyses revealed that VLPs structurally resemble the native virus but display distinct features in regions corresponding to epitopes that show differential antibody reactivity between VLP and IWV vaccines. By integrating structural and immunological analyses, we established a mechanistic framework linking capsid architecture to vaccine-induced antibody specificity. These findings suggest that VLP is a promising EV-D68 vaccine antigen with distinct epitope recognition profiles driven by structural characteristics.
2026-06-03 | Neutralizing antibodies elicited in nonhuman primates by an enterovirus D68 virus-like particle vaccine target receptor binding sites.
Enterovirus D68 (EV-D68) is a picornavirus that causes biennial outbreaks of respiratory disease in young children that can progress to rare but severe complications, including acute flaccid myelitis (AFM). EV-D68 virus-like particles (VLPs) elicit potent neutralizing antibodies that are protective in animal models. Here, we report the isolation and characterization of monoclonal antibodies elicited by EV-D68 VLPs in nonhuman primates (NHPs). We identified five potently neutralizing mAbs targeting overlapping epitopes near the capsid fivefold axis of symmetry formed by pentamers of viral protein 1. Cryo-electron microscopy structures of mAbs 1E11 and 5H03 in complex with VLP revealed epitopes on the capsid that bridge the sialic acid binding site and the proteinaceous entry receptor major facilitator superfamily domain-containing protein 6 binding site. We further characterized the mechanisms by which these mAbs neutralize EV-D68 and found that mAbs can disrupt multiple steps in the EV-D68 life cycle, including promoting premature uncoating. Antibodies elicited by VLP immunization of NHP protected as well as a best-in-class human mAb in a mouse challenge model, although single-amino acid mutations in the capsid enabled viral escape. Our results demonstrate that EV-D68 VLP-elicited mAbs target major viral sites of vulnerability, provide insight into their mechanisms of neutralization, and reinforce the potential for VLP-based vaccines as a countermeasure for EV-D68.
2026-05-06 | Propagation and immunological characterization of three enterovirus D68 strains using serum-free HEK293A suspension cell culture.
Enterovirus D68 (EV-D68) is an emerging pathogen that typically causes respiratory illness but can also lead to acute flaccid myelitis, which has been reported in several outbreaks worldwide. As EV-D68 has been found to be difficult to infect and proliferate in the standard Vero cells, a viable manufacturing process for the development of inactivated EV-D68 vaccine is urgently needed. We performed cell line screening and found that the HEK293A is a suitable host for EV-D68 propagation. The EV-D68 TW2014 (B3) strain was used to develop the EV-D68 production using a serum-free HEK293A (sfHEK293A) suspension system. Two major EV-D68 particles, the empty and full viral particles, were separated by ultracentrifugation and were found to induce low and high neutralizing antibody responses, respectively, in animal studies. To facilitate the mass production of vaccine, a combination of size-exclusion and anion-exchange chromatography was developed to purify EV-D68 particles. High quality, pure EV-D68 particles could be obtained by this simple procedure for vaccine production. Additionally, we found that the EV-D68 MO (B1) and KY (A2) strains could also be propagated in this sfHEK293A cell system, but at a lower 33 °C condition. These three formalin-inactivated EV-D68s were shown to induce EV-D68-specific neutralizing responses and also neutralize a recently circulating strain, but failed to cross-neutralize enterovirus A71 (EV-A71) in sera assay. The combination of EV-D68 and EV-A71 in one vaccination induced the neutralizing responses against both viruses. Our results showed that EV-D68 particles produced from the sfHEK293A suspension culture could be used to prepare EV-D68 vaccine candidates. Purifying the full EV-D68 particles for vaccine production is recommended for inducing a high, specific immune response. Combination of EV-D68 and EV-A71 is beneficial for the development of bivalent enterovirus vaccine.
2025-07-15 | Immunoinformatic Based Designing of Immune Boosting and Nonallergenic Multi-epitope Subunit Vaccine Against the Enterovirus D68.
Enterovirus D68 (EV-D68) is a non-enveloped, positive-sense, singlestranded RNA virus known for causing severe respiratory illnesses and its association with acute flaccid myelitis (AFM) in children. Despite its increasing public health significance, no vaccines or antiviral drugs are currently available for EV-D68. This study aimed to design an immune-boosting multi-epitope subunit vaccine against EV-D68 using advanced immunoinformatic and machine learning approaches. Capsid proteins VP1, VP2, and VP3 of EV-D68 were screened for immunogenic HTL, CTL, and B-cell epitopes to develop a non-allergenic, highly immunogenic multi-epitope vaccine. Predicted epitopes were subjected to 3D structural modeling and molecular dynamics simulations to validate folding and structural stability. Molecular docking and immune simulation techniques were employed to evaluate vaccine-TLR3 interactions and predict immune responses, respectively. Molecular docking analysis revealed strong binding affinities between the vaccine constructs and the TLR3 receptor, with scores of -299 kcal/mol, -361 kcal/mol, -258 kcal/mol, and -312 kcal/mol for VP1, VP2, VP3, and combined vaccine-TLR3 complexes. Molecular dynamic simulation and dissociation constant analyses confirmed the strength of these interactions, with binding free energies ranging from -57.75 kcal/mol to -101.35 kcal/mol. Codon adaptation index (CAI) values of 0.96 and GC content of ~69% supported the high expression potential of the vaccine constructs. Immune simulations demonstrated robust immune responses characterized by elevated IgG, IgM, cytokines, and interleukins, along with effective antigen clearance. The strong molecular interactions with TLR3 and simulated immune responses suggest that the designed vaccines can activate both innate and adaptive immunity. The high CAI and GC values support their expression feasibility in E. coli, enhancing prospects for production. This study provides a strong foundation for the development of a safe and effective EV-D68 vaccine, showcasing the potential of computational vaccine design.
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2025-11-18 | Efficacy and Safety of Plasmapheresis in Children With Acute Transverse or Flaccid Myelitis, and Guillain-Barré Syndrome.
Acute transverse myelitis (ATM), acute flaccid myelitis with polio-like myeloradiculitis (AFM) and Guillain-Barré syndrome (GBS) are severe neuroimmune disorders associated with high morbidity. First-line treatments include corticosteroids and intravenous immunoglobulins (IVIg), but they may be insufficient. Therapeutic plasma exchange (TPE) and immunoadsorption (IA) are second- or third-line options in pediatrics, despite their recognized efficacy in adults. Data on pediatric use remain limited. This study aimed to evaluate the efficacy and safety of apheresis in children with ATM, GBS or AFM. We conducted a retrospective monocentric study at Necker University Hospital, including pediatric patients diagnosed with ATM, GBS or AFM who underwent TPE/IA between 2014 and 2024. Functional outcomes were assessed using the modified Rankin Scale (mRS) at five time points, from peak disease severity to last follow-up. Safety was evaluated based on severe adverse events. Among 23 children (6 years [Q1-Q3]), 20 received TPE and 3 IA. 17 of 23 patients (74%) showed significant improvement by the end of treatment. The median mRS was 5 (IQR 4-5) before apheresis, improving to 4 (IQR 2-4) at the end of TPE/IA, and decreasing to 2 at 6 months. Brainstem function fully recovered in all surviving patients. Among 14 children with sphincter dysfunction, 5 (36%) still required intermittent catheterization. No treatment discontinuations occurred, but two patients died, one from venous air embolism, one after life-sustaining treatment limitation. TPE and IA appear effective in pediatric ATM, AFM and GBS. Larger studies are needed to confirm long-term efficacy and refine treatment guidelines.
2024-03-23 | Transcription factor EB (TFEB) interaction with RagC is disrupted during Enterovirus D68 Infection
Abstract Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is essential for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells, but not in autophagy defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. Importance Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis (AFM). The latter disease is associated with multiple non-polio enterovirus species, including Enterovirus D68 (EV-D68), Enterovirus 71 (EV-71), and coxsackievirus B3 (CVB3). Here we demonstrate that EV-D68 interacts with a host transcription factor, TFEB, to promote vRNA replication and regulate egress of virions from cells. TFEB was previously implicated in viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here we show EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically-important viruses differently.
2024-02-29 | Human B cells and dendritic cells are susceptible and permissive to enterovirus D68 infection.
Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.
2023-09-04 | Contemporary enterovirus-D68 isolates infect human spinal cord organoids.
Enterovirus D68 (EV-D68) is a nonpolio enterovirus associated with severe respiratory illness and acute flaccid myelitis (AFM), a polio-like illness causing paralysis in children. AFM outbreaks have been associated with increased circulation and genetic diversity of EV-D68 since 2014, although the virus was discovered in the 1960s. The mechanisms by which EV-D68 targets the central nervous system are unknown. Since enteroviruses are human pathogens that do not routinely infect other animal species, establishment of a human model of the central nervous system is essential for understanding pathogenesis. Here, we describe two human spinal cord organoid (hSCO)-based models for EV-D68 infection derived from induced, pluripotent stem cell (iPSC) lines. One hSCO model consists primarily of spinal motor neurons, while the another model comprises multiple neuronal cell lineages, including motor neurons, interneurons, and glial cells. These hSCOs can be productively infected with contemporary strains, but not a historic strain, of EV-D68 and produce extracellular virus for at least 2 weeks without appreciable cytopathic effect. By comparison, infection with hSCO with another enterovirus, echovirus 11, causes significant structural destruction and apoptosis. Together, these findings suggest that EV-D68 infection is not the sole mediator of neuronal cell death in the spinal cord in those with AFM and that secondary injury from the immune response likely contributes to pathogenesis. IMPORTANCE AFM is a rare condition that causes significant morbidity in affected children, often contributing to life-long sequelae. It is unknown how EV-D68 causes paralysis in children, and effective therapeutic and preventative strategies are not available. Mice are not native hosts for EV-D68, and thus, existing mouse models use immunosuppressed or neonatal mice, mouse-adapted viruses, or intracranial inoculations. To complement existing models, we report two hSCO models for EV-D68 infection. These three-dimensional, multicellular models comprised human cells and include multiple neural lineages, including motor neurons, interneurons, and glial cells. These new hSCO models for EV-D68 infection will contribute to understanding how EV-D68 damages the human spinal cord, which could lead to new therapeutic and prophylactic strategies for this virus.
2023-01-20 | RNA Structure in the 5′ Untranslated Region of Enterovirus D68 Strains with Differing Neurovirulence Phenotypes
Enterovirus-D68 (EV-D68) is a positive-sense single-stranded RNA virus within the family Picornaviridae. EV-D68 was initially considered a respiratory virus that primarily affected children. However, in 2014, EV-D68 outbreaks occurred causing the expected increase in respiratory illness cases, but also an increase in acute flaccid myelitis cases (AFM). Sequencing of 2014 outbreak isolates revealed variations in the 5′ UTR of the genome compared to the historical Fermon strain. The structure of the 5′ UTR RNA contributes to enterovirus virulence, including neurovirulence in poliovirus, and could contribute to neurovirulence in contemporary EV-D68 strains. In this study, the secondary and tertiary structures of 5′ UTR RNA from the Fermon strain and 2014 isolate KT347251.1 are analyzed and compared. Secondary structures were determined using SHAPE-MaP and TurboFold II and tertiary structures were predicted using 3dRNAv2.0. Comparison of RNA structures between the EV-D68 strains shows significant remodeling at the secondary and tertiary levels. Notable secondary structure changes occurred in domains II, IV and V. Shifts in the secondary structure changed the tertiary structure of the individual domains and the orientation of the domains. Our comparative structural models for EV-D68 5′ UTR RNA highlight regions of the molecule that could be targeted for treatment of neurotropic enteroviruses.
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